PROJECT ABSTRACT Many of the details of organelle-to-nucleus retrograde signaling pathways have been elucidated in yeast, mammalian and plant model systems. Understanding mechanisms of chloroplast-to-nucleus signaling will help elucidate the functions of retrograde signaling in all organisms including Bacteria and Apicomplexan. Our previous work with mutants lacking the chloroplast RNA helicase ISE2 indicates that chloroplasts are important regulators of plant intercellular communication and trafficking mediated by pores in the plant cell walls called plasmodesmata (PD). In the chloroplast, the methyl-D- erythritol 4-phosphate (MEP) pathway that produces isoprenoids is also the source of methylerythritol cyclodiphosphate (MEcPP), an important chloroplast-to-nucleus signaling metabolite. MEcPP signaling activates the biosynthetic pathway for a group of sulfur-containing plant secondary metabolites, glucosinolates. Our recent data suggests that glucosinolates or glucosinolate-derived molecules can regulate intercellular trafficking via PD. In this proposal we seek to examine the link between chloroplast-to-nucleus signaling via MEcPP and PD regulation. The specific aims of this proposal will 1) determine if glucosinolates or glucosinolate breakdown products modulate intercellular trafficking via PD, and 2) reveal if the chloroplast RNA helicase ISE2 uses the MEP chloroplast-to-nucleus signaling pathway to control intercellular trafficking via PD. The research proposed here will significantly advance our understanding of chloroplast-to-nucleus signaling, how intercellular trafficking via PD is regulated and how the two are connected. The research will also facilitate the PI?s training to become an independent scientist by providing broad experiences in a variety of genetic, imaging and biochemical approaches. The skills gained should be transferrable to diverse areas of biomedical and biological sciences as a postdoc and then independent scientist. The research training will be accompanied by professional development activities to ensure success in the PI?s future career as a faculty member.